Method for producing L-carnitine from crotonobetaine using a two stage continuous cell-recycle reactor

ABSTRACT

L(−)-carnitine is synthesized from crotonobetaine, crotonobetaine salts or derivatives in an ecologically advantageous manner by immobilizing cells of  Escherichia coli  044 K74 on ceramics, glass beads or polyurethane disks in a two stage continuously operating cell recycle reactor containing a reaction medium. The medium preferably contains between 25 mM and 1 M crotonobetaine and at least 50 mM fumarate. Growing or resting cells of  E. coli  are retained in the reactor by micro or ultrafiltration membranes which are arranged as a flat membrane module or hollow fiber module. A first stage contains a reactor tank and a second stage contains an external recirculation loop connected to the tank for feeding the reaction medium through a filter unit. L-carnitine is synthesized under anaerobic conditions to produce a reaction medium containing L-carnitine and unreacted crotonobetaine. The reaction medium is transferred through the recirculation loop to the filter unit to produce a filtrate containing L-carnitine and a residue containing unreacted crotonobetaine and cells. The residue is recirculated to the reactor tank.

FIELD OF THE INVENTION

The invention relates to a process for producing L-carnitine fromcrotonobetaine, from salts of crotonobetaine, other derivatives ofcrotonobetaine or the like.

BACKGROUND OF THE INVENTION

It is known that L-carnitine, a ubiquitously occurring compound, playsan important role in metabolism, especially in transporting long-chainfatty acids through the inner mitochondrial membrane. Numerous clinicalapplications derive from the function of carnitine in the metabolism ofeukaryotes, e.g., in the treatment of patients with carnitine deficiencysyndromes, in the prevention and therapy of various heart diseases andin the treatment of hemodialysis patients. Further, L-carnitine issignificant as a supplemental nutrient and also promotes, as an additiveto fermentation media, the growth of yeasts and bacteria. The growingneed for this biologically active L-carnitine enantiomer for these andother applications has led to a worldwide search for means ofsynthesizing this betaine in an optically pure form, since thechemically synthesized racemate cannot be used because it inhibitscarnitine acetyl transferase and the carnitine carrier protein.

To isolate the L-isomer, up to now processes have been used that arebased on splitting racemates by fractionated crystallization usingoptically active acids (e.g., U.S. Pat. No. 4,254,053, 1981), whereD(+)-carnitine occurs as a waste product.

This problem can be overcome by various biological processes, startingwith inexpensive achiral precursors (Adv. Biochem. Eng. Biotechnol.,1993, 50, 21-44) Of particular interest is stereospecific hydration oftrans-crotonobetaine into L-carnitine using strains of the generaEscherlchia (ED 0121444, 1984; DD 221 905, 1987; EP 0320460, 1989) orProteus (Agric. Biol. Chem., 1988, 52, 2415-2421; U.S. Pat. No.5,300,430, 1994) The advantage of this method lies in the fact that thisachiral precursor can also be obtained by chemical dehydration of thewaste product D-carnitine.

The numerous processes described in the literature with immobilizedmicroorganisms in a continuously operating reactor system have theadvantage that

pure reaction media can be used, thus facilitating the extraction andpurification process,

by using higher concentrations of the biocatalyst in the reactionmedium, higher productivities are achieved while the possibility ofcontamination is reduced,

there is reduced sensitivity to inhibitors or a nutrient deficiency,

a higher stability of the biocatalyst is achieved.

The advantages mentioned can also be applied to a commercially usedprocess.

A continuously operating reactor in which microorganisms are retained bymicro- or ultrafiltration membranes is an immobilization process which,besides the above-mentioned advantage, also entails lower costs for theimmobilization while making it possible to have a very slight upscaling.

SUMMARY OF THE INVENTION

Consequently the object of the invention is a process for producingL-carnitine from crotonobetaine, crotonobetaine salts or othercrotonobetaine derivatives in a continuous reactor with free orimmobilized cells, growing or resting Escherlchla coil 044K74 (DSM 8828)cells, that are retained by micro- or ultrafiltration membranes in aflat membrane or hollow fiber module.

DETAILED DESCRIPTION OF THE INVENTION

E. coil is kept in the reactor mentioned at temperatures between 20 and40° C., pH values between pH 6.0 8.0 and under anaerobic conditions thatare necessary for the induction of the enzyme that metabolizescarnitine.

A minimal or complex medium is used as the reaction medium. In bothcases, crotonobetaine, crotonobetaine salts or other crotonobetainederivatives are added in concentrations between 25 mmol and 1M. Theminimal medium contains varying concentrations of caseine hydrolyzateand salts (NH₄)₂50₄, KH₂PO₄, K₂HPO₄, MgSO₄x7H₂0, MnSO₄x4H₂0, FeSO₄x7H₂0,while the complex medium contains varying concentrations of pancreaticpeptone and NaCl. To improve the growth of E. coil, glycerine, glucose,ribose, saccharose or lactose are added. Also added to the medium areinhibitors that prevent the transformation of crotonobetaine intoy-butyrobetaine (fumarate, glucose or nitrate) and inductors ofcarnitine-metabolizing enzymes such as D-, L-, DL-carnitine, their saltsand derivatives or crotonobetaine, its salts or derivatives.

The course of the reaction in the continuous cell-recycle reactor usedhere can be divided into two stages. The one stage consists of a reactortank in which cells of E. coil, together with the reaction medium,convert most of the crotonobetaine into L-carnitine. This reactor tankhas monitoring elements for pH value, temperature and stirring speed andfor the monitoring and correction of oxygen concentration. The feed ofthe reaction medium into the reactor is performed with a dosing pump.When necessary, excess medium must be removed from the reactor tank. Thesecond stage consists of an external recycling loop that is connected tothe reactor tank and conveys the contents of the reactor through afilter unit by means of a pump. While the filtrate is being collected,to isolate L-carnitine from it as the reaction product, the residue fromthe filtration is fed again to the reactor. For filtering the cellsuspension, commercial filter systems of varying provenance can be usedas long as they have a pore size below the cell size of E. coil. Thespeed of the recycling pump remains unchanged to achieve the bestpossible filtration rates and to minimize the formation of apolarization membrane during the filtration process. Filtering may beperformed using commercial cross current filtration or hollow-fibermodules consisting of ultra-or microfiltration membranes composed ofcellulose, polysulphone or polysulphonated polysulphone with a retentionlimit of 300 kDa or 0.211 μ. The continuous cell-recycle reactor may beoperated at different levels of dilution adjusted by dosing andfiltration pumps, and at different agitation speeds and differentbiomass concentrations. Rate of discharge from the filtration pump iscontrolled by process control means.

The expression free E. coil cells indicates the state in which wholecells are suspended in the reaction medium without preventing a celloutflow through the exit solution. The expression immobilized cellsdescribes the state in which whole cells are bonded to soluble polymersor insoluble carriers, or are enclosed in membrane systems (in Methodsin Enzymol. 1987, vol. 135, 3-30)

The concept growth conditions is defined as the situation in which wholecells use substrates and form products during their life cycle. Restingcells are understood as intact cells that are not growing and that show,under certain conditions, special metabolic functions (in“Biotechnology” (Kieslich, K.; Eds. Rehm, N.J. and Reed, G.) VerlagChemie, Weinheim, Germany. 1984, Vol. 6a, 5-30)

The process is described below with several embodiments:

EXAMPLE 1

Escherlchla coil 044 K74 is cultivated in an Erlenmeyer flask that isfilled to the top and sealed air-tight at 37° C. under anaerobicconditions, on a rotating shaker (150 r.p.m.). The complex medium usedhas the following composition: 50 mM of crotonobetaine, 50 muM fumarate,5 g/l of NaCi and varying concentrations (between 0.5 and 10 g/l) ofpancreatic peptone. The pH is set using KOH to pH 7.5. Table 1summarizes the specific growth rates at varying peptone concentrations.

TABLE 1 Maximum specific growth rates of Escherlchla coil 044 K74Peptone (g/l) 0.5 1.0 2.5 5.0 10.0 pmax (h˜) 0.224 0.296 0.351 0.3720.325

Under the conditions described, growing cells of E. coil are able toproduce 20-30 mM of L-carnitine up to the end of the test.

Concentrations higher than 5 g/l of peptone yield similar growth andkinetic parameters as well as biomass content (OD 600 nm) In contrast,at lower peptone concentrations, lower growth parameters are obtained.

EXAMPLE 2

Escherlchla coil 044 K74 is cultivated in an Erlenmeyer flask that isfilled to the top and sealed air-tight at 37° C. under anaerobicconditions, on a rotating shaker (150 r.p.m.) The complex medium usedhas the following composition: 50 muM of crotonobetaine, 5 g/l ofpancreatic peptone, S g/l of NaCl and graduated concentrations (between0 and 75 muM) of fumarate. The pH is set using KOR to pH 7.5.

Table 2 shows that adding fumarate causes higher growth rates of E. coil044 K74 and an OD of 600 nm of almost 1.0 in the steady state. Further,fumarate causes L-carnitine formation of 20-30 muM up to the end of thetest. In the absence of fumarate, a carnitine concentration of only 5muM is obtained.

TABLE 2 Biomass (0D₆₀₀˜) at varying fumarate concentrations after a 10hour test. Fumarate (mM) 0 25 50 75 ˜max (h¹) 0.21 0.37 0.38 0.39Biomass OD (600 nm) 0.980 0.910 1.00 0.950

EXAMPLE 3

The ability of Escherlchla coil 044 K74 to form L-carnitine fromcrotonobetaine is induced by crotonobetaine. The induction studies wereperformed with crotonobetaine between 5 and 75 muM using resting cells.At higher crotonobetaine concentrations, conversion rates of above 60%of L-carnitine are achieved (Table 3)

TABLE 3 Production of L-carnitine by resting cells of Escherlchla coil044 K74 as a function of varying crotonobetaine concentrations.Crotonobetaine (mM) 5 25 50 75 L-carnitine 55 60 62 E5 production (%)

EXAMPLE 4

Escherlchla coil 044 K74 is cultivated in an Erlenmeyer flask that isfilled to the top and sealed air-tight at: 37° C. under anaerobicconditions, on a rotating shaker (150 r.p.m.). The complex medium usedhas the following composition: 50 mM of crotonobetaine, 5 g/l ofpancreatic peptone, 5 g/l of NaCl and 50 mM of fumarate. The pH is setusing KON to pH 7.5.

To raise the biocatalyst concentration in the reactor and to make itpossible to have L-carnitine production at dilution rates higher thanthe maximum specific growth rate, a membrane reactor was used. The cellswere retained with a polysulfone microfiltration membrane with anexclusion threshold of 0.1 pm and were used again. The membranes werearranged in a plate module. Table 4 summarizes the biomass content andTable 5 summarizes the carnitine production, the crotonobetaineconversion and the productivity in this system.

TABLE 4 Biomass content of E. coil 044 K74 in a continuously operatingmembrane reactor. Dilution rate (h¹) 0.0 0.2 1.0 2.0 Biomass(gdryweight/1) 0.5 2.1 9.4 27.0

TABLE 5 L-carnitine production, crotonobetaine conversion andproductivity in a continuously operating cell reactor wlih Escherlchlacoil 044 K74 Dilution rate (tf¹) 0.0 0.5 1.0 1.5 2.0 L-carnitine 0.0 3842 42 38 production (%) Crotonobetaine 0.0 24 26 26 30 conversion (%)Productivity 0.0 1.75 3.5 5.5 6.5 ˜

It can be seen from the tables that, with immobilized cells ofEscherlchla coil 044 K74 in a cell recycle reactor, 6.5 1/h ofL-carnitine was formed from crotonobetaine with a metabolization rate ofalmost 40%.

What is claimed is:
 1. A method for producing L-carnitine fromcrotonobetaine, comprising the steps of: (1) introducing between 25 mMand 1 M crotonobetaine or a salt or derivative thereof and at least 50mM fumarate into a reaction a two stage continuously operatingcell-recycle reactor wherein a first stage consists of a reactor tankcontaining the reaction medium and cells of a strain of the generaEscherichia that convert crotonobetaine to L-carnitine, said cells beingimmobilized on a carrier selected from ceramics, glass beads andpolyurethane disks, and a second stage consists of an externalrecirculation loop connected to the reactor tank, by means of which thecontents of the reactor are fed through a filter unit; (2) synthesizingthe L-carnitine under anaerobic condition in the reaction mediumcontaining fumarate and crotonobetaine to produce a reaction mediumcontaining L-carnitine and unreacted crotonobetaine; and (3)transferring said reaction medium through the recirculation loop to saidfilter unit where the reaction medium is filtered to produce a filtratecontaining the L-carnitine from the reaction medium and recirculatingfrom the filter unit a residue containing unreacted cronobetaine intothe reactor tank of step (1).
 2. A method according to claim 1, whereinthe cells are E. coli 044 K74 (DSM 8828).
 3. A method according to claim2, wherein the carrier does not impair viability of the cells.
 4. Amethod according to claim 1, wherein the cells are immobilized onceramics.
 5. A method according to claim 2, wherein the reaction mediumcontains 50 mM crotonobetaine, 5 g/l pancreatic peptone, 5 g/l NaCl and50 mM fumarate, has a pH of 7.5, and the cell-recycle reactor comprisesa continuously operating membrane reactor.
 6. A method according toclaim 1, wherein in step (3) the residue contains the cells and thecells are continuously recycled to the reactor tank, and the reactionmedium is filtered by cross-flow filtration or hollow fiber modulesconsisting of ultra- or microfiltration membranes.
 7. A method accordingto claim 6, wherein the membranes are composed of cellulose,polysulphone or polysulphonated polysulphone with a retention limit of300 kDa or 0.211μ.
 8. A method according to claim 1, wherein thecell-recycle reactor is operated at different levels of dilution whichare adjusted by a dosing pump and a filtration pump, and at differentagitation speeds and different biomass concentrations.
 9. A methodaccording to claim 8, wherein a rate of discharge of the filtration pumpis controlled by process control means.